The mechanism of the in vivo irreversible binding to tissue protein of (14C) chloramphenicol was investigated by determining the chemical nature of its bound metabolites. By employing several techniques such as Sephadex and tlc chromatography, chemical and enzyme hydrolysis, and reverse isotope dilution analysis, it was found that the majority of the (14C) bound material consisted of approximately 50 percent glycine and 50 percent serine. Nearly an identical result was obtained when the plasma protein from rats administered (14C) dichloroacetate acid was investigated. These findings establish that CAP and dichloroacetic acid are metabolized by a similar route to yield two carbon units. These fragments are converted into glycine and serine and subsequently incorporated into protein. This route of metabolism may represent minor but a unique detoxifying process since it appears to involve the hydrolytic cleavage of the chlorines from CAP. These chlorines have previously been shown to be structural requirements for the in vitro activation of CAP into a reactive acylating agent. BIBLIOGRAPHIC REFERENCE: Gillette, J.R., and Pohl, L.R.: A prospective on covalent binding and toxicity. J. Toxicol. Environ. Health 2: 849-871, 1977.